Targeting pulmonary epithelium using adrp

ABSTRACT

This invention provides novel compositions and methods for the specific and/or preferential delivery of an effector (e.g. a drug or label) to an epithelial cell (e.g. a pulmonary epithelium). The compositions comprise an adipocyte differentiation-related protein (ADRP) attached to an effector thereby forming a chimeric moiety. The chimeric moiety is preferentially delivered to epithelial cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. Ser. No. 11/397,320, filed onApr. 3, 2006 which claim benefit of and priority to U.S. Ser. No.60/668,418, filed on Apr. 4, 2005, which are both incorporated herein byreference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[Not Applicable]

FIELD OF THE INVENTION

This invention pertains to the field of oncology. In particular, thisinvention pertains to the discovery that adipocytedifferentiation-related protein (ADRP) can be exploited tospecifically/preferentially deliver an effector (e.g. a retinoic acid)to a cell comprising the pulmonary epithelium.

BACKGROUND OF THE INVENTION

The pulmonary lipofibroblast is located in the alveolar interstitiumwhere it is distinguished by the presence of large, cytoplasmic lipiddroplets (Heid et al. (1996) Biochem J., 320 (Pt 3):1025-1030; Torday etal. (1995) Biochim. Biophys. Acta., 1254(2): 198-206). These cells werefirst described by O'Hare and Sheridan in 1970 (Nicholas et al. J ApplPhysiol. 53(6): 1521-1528), and their biochemical and structuralcharacteristics were determined during the late 1970s and early 1980s byBrody's group (Heid et al. (1996) Biochem J., 320(Pt 3): 1025-1030;Maksvytis et al. (1984) J. Cell Physiol., 118(2):113-123; Maksvytis etal. (1981) Lab Invest. 45(3): 248-259; Torday et al. (1995) Biochim.Biophys. Acta., 1254(2):198-206), which named them lipid interstitialcells. McGowan and Torday (McGowan and Torday (1997) Annu. Rev. Physiol.59: 43-62) have recently critically reviewed the literature on thecontributions of these cells to alveolar development and have termedthem lipofibroblasts to highlight their fibroblast-like phenotype.Torday and colleagues (Miura et al. (2002) J. Biol. Chem. 277(35):32253-32257; Nunez and Torday (1995) J. Nutr. 125(6 Suppl): 1639S-1644S)have investigated the prenatal ontogeny of the fetal rat lunglipofibroblast, showing a four- to fivefold increase of triacylglycerolin isolated lipofibroblasts, paralleling that in whole lung (Shannon etal. (2001) Am. J. Respir. Cell Mol. Biol. 24(3): 235-244), over the last4 days of gestation. The triacylglycerol content of fetal rat lunglipofibroblasts is maximal just before the appearance of surfactantphospholipid-containing lamellar bodies in neighboring alveolar type IIepithelial (EPII) cells, the site of pulmonary surfactant synthesis(Rodriguez et al. (2001) Exp. Lung Res. 27(1): 13-24). Torday andcoworkers have demonstrated in a coculture system that thetriacylglycerols of fibroblast origin are used for surfactantphospholipid synthesis by EPII cells (Rubin et al. (2004) Dev. Dyn.230(2): 278-289; Torday et al (1995) BBA 1254(2): 198-206) and that themetabolism of these lipids in the culture system is regulated byhormones important for lung maturation (Miura et al. (2002) J. Biol.Chem. 277(35): 32253-32257; Nunez and Torday (1995) J. Nutr. 125(6Suppl): 1639S-1644S).

In most mammalian cells, neutral lipids, including those found inpulmonary lipofibroblasts (LIFs), are stored in discrete lipid storagedroplets, which are composed of a core of triacylglycerol andcholesterol esters surrounded by a limiting osmophilic boundary(Brasaemle et al. (1997) J. Lipid Res. 38(11): 2249-2263). Little isknown about the proteins that are present at the surface of these lipidstorage droplets. The first-described intrinsic lipid droplet-associatedproteins were the perilipins, which localize to the periphery of theintracellular neutral lipid storage droplets in adipocytes (Adamson etal. (1991) Exp. Lung Res. 17(4): 821-835; Gao and Serrero (1999) J.Biol. Chem. 274(24): 16825-16830; Laemmli (1970) Nature 227(259):680-685; O'Hare and Sheridan (1970) Am. J. Anat. 127(2): 181-205) andsteroidogenic cells of the adrenal cortex, testes, and ovaries (O'Hareand Sheridan (1970) Am. J. Anat. 127(2): 181-205). Perilipins sharesequence homology with adipocyte differentiation-related protein (ADrP),which was first identified as a gene expressed very early in adipocytedifferentiation (Heid et al. (1998) Cell Tissue Res., 294(2): 309-321).ADrP, transfected into COS cells, has been shown to play a role infacilitated fatty acid uptake (Frolov et al. (2000) J. Biol. Chem.275(17): 12769-12780). ADrP mRNA has subsequently been found to beexpressed in a wide variety of somatic tissues: heart, brain, spleen,liver, skeletal muscle, kidney, testes, and most pronouncedly in thelung (Brasaemle et al. (1997) J. Lipid Res. 38(11): 2249-2263; Laemmli(1970) Nature, 227(259): 680-685). The expression level of ADrP mRNA inadult mouse lung was found to be second only to that in adipose tissue,the tissue that stores the greatest amount of neutral lipid and has thehighest expression of ADrP mRNA (Brasaemle et al. (1997) J Lipid Res.38(11): 2249-2263). Schultz et al have determined that lipofibroblastsexpress ADRP, but that neighboring alveolar type II cells express littleif any ADRP. Furthermore, ADRP binds to type II cells, facilitatinguptake and incorporation of triglyceride into surfactant phospholipid.ADrP was also identified in human tissues and named adipophilin(Greenberg et al. (1993) Proc. Natl. Acad. Sci. USA., 90(24):12035-12039).

SUMMARY OF THE INVENTION

This invention pertains to novel methods and compositions for directingeffectors (e.g. drugs, labels, etc.) to lung epithelium and/or to thenucleus of cells comprising the lung epithelium. The compositions andmethods are particularly well suited to direct therapeutic retinoic acidderivatives or other labels or therapeutic moieties directly to the lungepithelium to detect, visualize, and/or to treat lung cancer.

We determined that the ADRP and/or a complex or chimeric moietycomprising ADRP complex binds to the epithelial cell surface and istransported to the nucleus of pulmonary epithelial cells and other cellsthat participates in the ADRP lipid trafficking mechanism describedherein.

This invention thus provides compositions and methods for transportingvarious effectors to lung tissue. The compositions and methods providedherein are thus useful to treat damaged lung epithelium in acute orchronic lung diseases (e.g., various lung cancers, chronic obstructivepulmonary disease, acute asthma, and the like).

Thus, in certain embodiments, this invention provides a compositioncomprising an isolated adipocyte differentiation-related protein (ADRP)covalently coupled to or complexed with an effector. In certainembodiments the effector comprises a lipid or liposome, and the lipid orliposome can be empty or can contain or be complexed with a therapeuticagent. In certain embodiments the ADRP is a full length ADRP. In certainembodiments the ADRP is a carboxyl terminal fragment of ADRP ofsufficient length to induce transport of the effector (e.g. a lipid orliposome, a cytotoxin, a chelate, etc.) to and/or into an epithelialcell of the lung tissue. In various embodiments lipid or liposome is aneutral lipid or a liposome formed of neutral lipids. In certainembodiments the lipid or liposome is a neutral lipid or a liposomecomprising triacylglycerol. In various embodiments the lipid or liposomecomprises an agent selected from the group consisting of a retinoid, aprostanoid, an anti-inflammatory agent, a growth factor, athiazolidinedione, a chemokine, a chemotherapeutic, and the like. Invarious embodiments the lipid or liposome is a multilamellar liposome ora unilamellar liposome.

In various embodiments this invention also provides a compositioncomprising an adipocyte differentiation-related protein (ADRP)covalently coupled to, or complexed with, a lipid or liposome, whereinsaid lipid is complexed with an effector or said liposome contains aneffector. In various embodiments the ADRP is a full length ADRP or afragment (e.g. at least 30, 40, or 50 aa, preferably at least 80, 100,or 150 aa, more preferably at least 200, 250, or 300 aa, and mostpreferably at least 350 or 400 aa) of an ADRP (e.g., a carboxyl terminalfragment of ADRP of sufficient length to induce transport of the lipidor liposome to or into an epithelial cell of the lung tissue. In certainembodiments the lipid or liposome is a neutral lipid or a liposomeformed of neutral lipids that can optionally comprise triacylglycerol.In certain embodiments the lipid or liposome is a multilamellar or aunilamellar liposome. In certain embodiments the effector a label, acytotoxin, a drug, a prodrug, a cytokine, and the like. When theeffector is a cytotoxin, suitable cytotoxins include, but are notlimited to a Diphtheria toxin, a Pseudomonas exotoxin, a ricin, anabrin, and a thymidine kinase. When the effector is a detectable label,suitable detectable labels include, but are not limited to a radioactivelabel, a spin label, a colorimetric label, a fluorescent label, and aradio-opaque label. In certain embodiments the label is an isotopeselected from the group consisting of ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As,¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ⁵²Mn, ⁵¹Cr, ¹⁸⁶, Re, ¹⁸⁸Re,⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb,¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb,¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and ¹¹¹Ag. In various embodiments the effectorcomprises an alpha emitter. In various embodiments the effector is adrug selected from the group consisting of retinoic acid, a retinoicacid derivative, doxirubicin, vinblastine, vincristine,cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, a camptothecinderivative, interferon, tamoxifen, and taxol.

Also provided is a method of visualizing a tissue comprising ADRPreceptors, where the method involves: contacting the tissue with acomposition comprising an adipocyte differentiation-related protein(ADRP) covalently coupled to a detectable label (e.g., coupled to orcomplexed with, a lipid or liposome, wherein said lipid is complexedwith a detectable label or the liposome contains a detectable label);and detecting the label in the tissue. In certain embodiments the tissuecomprises a small cell carcinoma. In various embodiments, the ADRP is afull length ADRP or an ADRP fragment (e.g., as described above). Incertain embodiments the lipid or liposome can include any of the lipidsor liposomes described above. In certain embodiments the label isselected from the group consisting of a radioactive label, a spin label,an NMR label, and a radio-opaque label. In certain embodiments thedetecting comprises a method selected from the group consisting of anNMR scan, a CAT scan, a PET scan, and an X-ray.

Also provided is a method of inhibiting the growth or proliferation of atumor cell, where the method involves contacting the tumor cell with acomposition comprising an adipocyte differentiation-related protein(ADRP) covalently coupled to, or complexed with an effector comprising acancer therapeutic (e.g. a lipid or liposome, wherein said lipid iscomplexed with, or said liposome contains, a cancer therapeutic). Incertain embodiments the cancer therapeutic is selected from the groupconsisting of retinoic acid, a retinoic acid derivative, doxirubicin,vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin,5-fluorouracil, a camptothecin derivative, interferon, tamoxifen, andtaxol. In various embodiments ADRP is a full-length (human) ADRP or anADRP fragment (e.g., as described above). In certain embodiments thecancer comprises a small cell carcinoma.

This invention also provides a method of preferentially delivering aneffector to a cell comprising a pulmonary epithelial cell (e.g., a tumorcell in a mammal), where the method involves providing said effector ina liposome or complexed with a lipid, wherein said lipid or saidliposome are complexed with or covalently attached to an adipocytedifferentiation-related protein (ADRP); administering said lipid orliposome to said mammal whereby said lipid or liposome is preferentiallyinternalized by said pulmonary epithelial cell. In various embodimentsthe ADRP includes a full length ADRP or an ADRP fragment (e.g., asdescribed above) and the lipid or liposome comprises a lipid or liposomeas described above. In certain embodiments the lipid or liposomecomprises an agent selected from the group consisting of a retinoid, aprostanoids, an anti-inflammatories, a growth factor, athiazolidinediones, a chemokine, and a chemotherapeutic. In certainembodiments the pulmonary epithelia cell comprises a small cellcarcinoma. In various embodiments the effector is selected from thegroup consisting of a label, a cytotoxin, a drug, a prodrug, and acytokine @ilce the effector is a cytotoxin selected from the groupconsisting of a Diphtheria toxin, a Pseudomonas exotoxin, a ricin, anabrin, and a thymidine kinase. In certain embodiments the effector is adetectable label selected from the group consisting of a radioactivelabel, a spin label, a colorimetric label, a fluorescent label, and aradio-opaque label. In certain embodiments the effector comprises anisotope selected from the group consisting of ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ¹¹¹In, ^(113m)In, ₉₇Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ⁵²Mn, ⁵¹Cr, ¹⁸⁶, Re,¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au,¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm,¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh and ¹¹¹Ag. In certain embodiments theeffector comprises an alpha emitter. In certain embodiments the effectorcomprises a drug selected from the group consisting of retinoic acid, aretinoic acid derivative, doxirubicin, vinblastine, vincristine,cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, a camptothecinderivative, interferon, tamoxifen, and taxol.

DEFINITIONS

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers. The term also includes variants on the traditionalpeptide linkage joining the amino acids making up the polypeptide.

The phrase “specifically deliver”, as used herein, refers to thepreferential association of a molecule, or other moiety, with a cell ortissue bearing a particular target (e.g., receptor, ligand, etc.) ormarker as compared to cells or tissues lacking that target target ormarker. It is, of course, recognized that a certain degree ofnon-specific interaction may occur between the moiety and a non-targetcell or tissue. Nevertheless, specific delivery, may be distinguished asmediated through specific recognition of the target. Typically specificdelivery results in a much stronger association between the deliveredmoiety and cells bearing the target than between the moiety and cellslacking the target. In certain embodiments specific delivery typicallyresults in greater than 2 fold, preferably greater than 5 fold, morepreferably greater than 10 fold and most preferably greater than 100fold increase in amount of delivered moiety (per unit time) to a cell ortissue bearing the target as compared to a cell or tissue lacking thetarget or marker.

A “chimeric moiety” or “chimeric structure” refers to a moiety in whichtwo or more moieties (e.g., molecules) that exist separately in theirnative state are joined together to form a single moiety having thedesired functionality of all of its constituent components.

The terms “effector” or “effector component” refers to a moiety that isto be specifically and/or preferentially transported to the target towhich the chimeric moiety is directed. The effector typically has acharacteristic activity that is desired to be delivered to the target.Effector molecules include, but are not limited to drugs, liposomes,cytotoxins, labels, radionuclides, ligands, antibodies, and the like.

The term “targeting moiety” or “targeting component” refers to acomponent of a chimeric moiety that specifically and/or preferentiallytargets a particular cell or cell type. Thus for example, an ADRPtargeting moiety refers to a moiety that specifically and/orpreferentially binds to or associates with a cell expressing an ADRPreceptor. In certain embodiments, the ADRP targeting moiety refers to amoiety (e.g. ADRP, an ADRP fragment, etc.) that participates in the ADRPlipid trafficking mechanism described herein.

The term “residue” as used herein refers to an amino acid that isincorporated into a polypeptide. The amino acid may be a naturallyoccurring amino acid and, unless otherwise limited, may encompass knownanalogs of natural amino acids that can function in a similar manner asnaturally occurring amino acids.

A “fusion protein” refers to a polypeptide formed by the joining of twoor more polypeptides through a peptide bond formed between the aminoterminus of one polypeptide and the carboxyl terminus of anotherpolypeptide and/or through a peptide linker. The fusion protein can beformed by the chemical coupling of the constituent polypeptides or itcan be expressed as a single polypeptide, e.g., from nucleic acidsequence encoding the single contiguous fusion protein. A single chainfusion protein is a fusion protein having a single contiguouspolypeptide backbone.

A “spacer” or “peptide linker” as used herein refers to a peptide thatjoins the proteins comprising a fusion protein. Generally a spacer hasno specific biological activity other than to join the proteins or topreserve some minimum distance or other spatial relationship betweenthem. However, the constituent amino acids of a spacer can be selectedto influence some property of the molecule such as the folding, netcharge, or hydrophobicity of the molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the time-course and intracellular localization of theGFP-ADRP lipid complexes in cultured A549 cells. Cultured A549 cellswere incubated with GFP-ADRP lipid droplets and examined at 0, 10 min, 2h, and 24 h by con-focal microscopy. Although there were no visibledroplets at the baseline (0 min), there was a rapid uptake and transitof the GFP-ADRP complex to the perinuclear region at 10 min. At 2 h,these complexes were localized to both perinuclear and cytoplasmiccompartments, and at 24 h, the GFP-ADRP complexes were more diffuselyspread throughout the cytoplasm.

FIG. 2 shows the dose-response and intracellular localization of theGFP-ADRP lipid complexes in cultured A549 cells. Cultured A549 cellswere incubated with no added LDs (0 μg/ml, 50 μg/ml or 100 μg/ml mediumfor 2 h, and then examined by con-focal microscopy. There were no cellsshowing GFP-ADRP lipid complexes without added LDs, a few with 50 μg/mlLD, and markedly increased GFP-ADRP perinuclear complexes with 100 μg/mlLD (see arrows).

FIG. 3 shows that the uptake of ADRP by A549 cells induces SP-B mRNAexpression: Cultured A549 cell monolayers were treated with 0, 10, 50,or 100 μg/ml GFP-ADRP lipid complexes for 24 h, and then SP-B mRNAexpression was examined by RT-PCR. Note the step-wise increase in SP-BmRNA expression over the dosage range used, resulting in an 80% increaseat the highest LD dose (100 ng/ml) (n=3; *, p<0.05 vs controls byAnalysis of Variance).

FIG. 4 shows that actinomycin D inhibition of ADRP-induced SP-B mRNAexpression. Actinomycin D (1 μg/ml) blocked ADRP induction of SP-B mRNAexpression by A549 cells (n=3; *, p<0.05 vs without actinomycin byunpaired t test).

FIG. 5. ADRP increases SP-B protein levels in A549 cells: A549 cellmonolayers were treated with 0, 10, 50, or 100 μg/ml GFP-ADRP lipidcomplexes for 24 h and SP-B levels were subsequently determined byWestern blot hybridization. Note the step-wise increase in SP-B proteinlevels over the dosage range used, resulting in a 50% increase at thehighest LD dose (100 ng/ml) (n=3; *, p<0.05 vs controls by Analysis ofVariance).

FIG. 6 shows that cycloheximide inhibits ADRP-LD-stimulated SP-B proteinincrease. Concomitant treatment of A549s incubated with ADRP withcycloheximide (5 :g/ml) inhibited the increase in SP-B protein levels(n=3; *, p<0.05 vs without actinomycin by unpaired t test).

FIG. 7 shows that uptake of ADRP-LDs stimulates surfactant phospholipidsynthesis: Incubation of A549 cells with graded doses of ADRP-LDs (100:g/ml) for 24 h stimulated saturated phosphatidylcholine synthesis57-fold. Co-incubation of these ADRP-LD-exposed A549 cells with gradeddoses of ADRP antibody (0.1, 0.4, 2 :g/ml) showed a dose-dependentinhibition of ADRP-LD-induced saturated phosphatidylcholine synthesis(M, p<0.05 vs control; ψ, p<0.001 vs control). Neither preimmune serumnor a non-specific IL-6 antibody showed the inhibitory effect of ADRP-LDeffect on saturated phosphatidylcholine synthesis, indicating thespecificity of the ADRP antibody effect.

FIG. 8 illustrates uptake of GFP-ADRP LDs in vivo. In vivoadministration of graded doses of ADRP-LDs (0, 45, 450, or 4500 :g/kg)to ventilated adult rats resulted in a dose-dependent increase inSurfactant Protein-B expression by the lung 30 minutes after injection.(n=3, *, p<0.05 vs control=time 0).

FIG. 9 illustrates a schematic for the lipid trafficking mechanism forcoordinate regulation of surfactant protein and phospholipid synthesis,which depicts (1) the active recruitment of circulating lipid by thelipofibroblast, (2) the formation of ADRP-lipid droplets by thelipofibroblast, (3) active ADRP-LD secretion (4) in response tostretch-regulated, alveolar type II cell-produced PTHrP andprostaglandin E₂ (PGE₂), (5) ADRP-LD uptake by alveolar type II cellsvia a receptor-mediated mechanism, and (6) coordinate regulation ofsurfactant phospholipid and SP-B, resulting in simultaneous increases insurfactant protein and phospholipid production by the alveolar type IIcell.

DETAILED DESCRIPTION

This invention pertains to novel methods and compositions for directingeffectors (e.g. drugs, labels, etc.) to lung epithelium and/or to thenucleus of cells comprising the lung epithelium. The compositions andmethods are particularly well suited to direct therapeutic retinoic acidderivatives or other labels or therapeutic moieties directly to the lungepithelium to detect, visualize, and/or to treat lung cancer or damagedlung epithelium in acute or chronic lung diseases (e.g., chronicobstructive pulmonary disease, acute asthma, and the like).

Lipids and lipid associated substances such as retinoids are activelytaken up from the circulation by lung fibroblasts, which express theAdipocyte Differentiation-Related Protein (ADRP). ADRP is responsiblefor the uptake and storage of these lipid inclusions, which aretypically composed of triglycerides and retinoic acid. The neighboringepithelial cells secrete prostaglandin E₂, which causes the secretion ofthe ADRP lipid complexes by the fibroblasts. We determined that the ADRPcomplex binds to the epithelial cell surface and is transported to thenucleus, where it stimulates surfactant protein mRNA synthesis, probablydue to binding of retinoic acid to the promoter sequence of thesurfactant protein gene in the nucleus. Since this targeting mechanismdirects retinoids from the circulation to the lung epithelium, it can beexploited to deliver therapeutic retinoic acid derivatives or othermoieties directly to the epithelium to treat lung cancer.

In particular, we demonstrated that ADRP complexed totriglyceride/retinoic acid (RA) moves from the circulation tolipofibroblasts, and on to type II cells, where it enters the nucleus tostimulate SP-B mRNA expression. The net result is increased surfactantphospholipid and protein expression. Using green fluorescent protein(GFP)-labeled ADRP lipid droplets to study the transit of lipid dropletsthrough the type II pneumocyte to the nucleus we demonstrated that whenH441 cells were incubated with GFP-ADRP Lipid proplets extracted fromChinese Hamster Ovary cells for 10 minutes and subsequently examined byconfocal microscopy. GFP-ADRP localized to the nuclei of the H441 cells.In addition, GFP-ADRP LDs were administered to adult rats intravenously.Uptake of green fluorescent protein-adipocyte differentiation relatedprotein (GFP-ADRP) lipid droplets in vivo was demonstrated.

Without being bound to a particular theory, we believe the traffickingof lipid from the circulation to the epithelial cell nucleus has neverbeen described before. It is a novel route for the exposure of the lungepithelium to substrate or drugs.

Thus, liposomes coated with ADRP, containing all-trans retinoic acid, orother retinoic acid derivatives known to affect lung carcinomas, orvarious labels, therapeutic or other effectors can be used to deliverthe desired effector to the epithelium of the lung. In certainembodiments, the liposomes are into a vehicle (e.g., a pharmaceuticallyacceptable excipient) and dripped down the airway or injectedintravenously.

The methods need not be limited to the use of liposomes. ADRP proteinscan be coupled to essentially any moiety that it is desired topreferentially deliver to lung epithelium.

Thus, in certain embodiments, this invention provides for compositionsand methods for impairing the growth of tumors. The methods involveproviding a chimeric moiety comprising targeting moiety (e.g., ADRP oranother moiety that binds an ADRP receptor) attached to an effector. Theeffector can comprise retinoic acid, and/or other cancer therapeutic(e.g., vinblastine, doxorubicin, a cytotoxin such as Pseudomonasexotoxin (PE), Diphtheria toxin (DT), ricin, abrin, and the like).

The chimeric moiety is administered to an organism whereby the ADRPcomponent causes preferential and/or specific delivery to the targettissue (e.g. pulmonary epithelia).

The use of chimeric moieties comprising a targeting moiety joined to aneffector to target tumor cells has been described. For example, chimericfusion proteins which include interleukin 4 (IL-4) or transforminggrowth factor (TGFα) fused to Pseudomonas exotoxin (PE) or interleukin 2(IL-2) fused to Diphtheria toxin (DT) have been tested for their abilityto specifically target and kill cancer cells (Pastan et al. (1992) Ann.Rev. Biochem., 61: 331-354).

In certain embodiments, this invention also provides for compositionsand methods for visualizing and/or detecting the presence or absence oftarget cells (e.g., tumor cells expressing an ADRP receptor). Thesemethods involve providing a chimeric moiety comprising an effector, thatis a detectable label attached to a targeting moiety (e.g., ADRP orother moiety that specifically binds an ADRP receptor). The ADRPreceptor targeting moiety specifically binds the chimeric moiety to thetarget cells which are then marked by their association with thedetectable label.

In certain embodiments, the effector can be another specific bindingmoiety such as an antibody, a growth factor, or a ligand. The chimericstructure then acts as a highly specific bifunctional linker. Thislinker may act to bind and enhance the interaction between cells orcellular components to which the chimeric moiety binds. Thus, forexample, where the “targeting” component of the chimeric moleculecomprises a polypeptide (e.g., ADRP) that specifically binds to an ADRPreceptor and the “effector” component is an antibody or antibodyfragment (e.g. an Fv fragment of an antibody), the targeting componentspecifically binds, e.g., cancerous pulmonary epithelial cells, whilethe effector component binds receptors (e.g., IL-2 or IL-4 receptors) onthe surface of immune cells. The chimeric moiety can thus act to enhanceand direct an immune response toward the target cells.

As indicated above, the effector can comprise one or morepharmacological agents (e.g., a drugs) and/or a vehicle comprising apharmacological agent. This is particularly suitable where it is merelydesired to invoke a non-lethal biological response. Thus the moiety thatspecifically binds to an ADRP receptor may be conjugated to a drug suchas retinoic acid, a retinoic acid analogue or derivative, vinblastine,doxirubicin, genistein (a tyrosine kinase inhibitor), an antisensemolecule, and other pharmacological agents known to those of skill inthe art, thereby specifically targeting the pharmacological agent tocells expressing ADRP receptors.

In certain embodiments, the targeting component can be bound to avehicle containing the therapeutic composition. Such vehicles include,but are not limited to liposomes, micelles, various synthetic beads, andthe like.

One of skill in the art will appreciate that the chimericmoieties/structures of the present invention can include multipletargeting moieties (e.g., ADRPs) bound to a single effector orconversely, multiple effectors bound to a single targeting moiety. Instill other embodiment, the chimeric moieties can include both multipletargeting moieties and multiple effector molecules. Thus, for example,this invention provides for “dual targeted” cytotoxic chimeric moleculesin which targeting molecule that specifically binds to ADRP is attachedto a cytotoxic molecule and another molecule (e.g. an antibody, oranother ligand) is attached to the other terminus of the toxin.

The foregoing embodiments are meant to be illustrative and not limiting.Using the embodiments described herein, other suitable ADRPtargeting/effector constructs will be apparent to one of skill in theart.

I. Indications.

As indicated above, in certain embodiments, the chimeric moietiesdescribed herein are used to direct retinoic acid or other retinoids toa target tissue (e.g., pulmonary epithelia). Since retinoids are usefulin treating a wide variety of epithelial cell carcinomas-head, neck,esophagus, adrenal, prostate, ovary, testes, pancreas, gut this approachis expected to be useful for all of these life-threatening cancers.

We have also shown that lung fibrosis is due to molecular injury to theepithelium of the lung. Therefore, one can use the chimeric moieties ofthis invention to treat a wide variety of lung fibrotic diseasesincluding, but not limited to Bronchopulmonary Dysplasia, emphysema,asthma, chronic obstructive lung disease, idiopathic lung fibrosis, andthe like.

In addition, there are many chronic degenerative diseases that arecharacterized by epithelial cell toxicity. These include, but are notlimited to pancreatitis, kidney tubular disease, liver fibrosis,reperfusion injury of the vasculature, prostatitis. The chimericmoieties of this invention can be used to treat these conditions as wellsince all of these tissues utilize the ADRP lipid trafficking mechanism.

II. ADRP Chimeric Moieties.

As explained above, it was a surprising discovery that ADRP can be usedas a targeting moiety to specifically direct coupled effectors (e.g.,proteins or other moieties) to epithelial tissues, in particular topulmonary epithelium. In various embodiments this involves the use ofchimeric moieties/structures comprising a targeting molecule (e.g., anADRP) attached to an effector (e.g. a liposome, radionuclide, etc.). Thechimeric moieties of this invention specifically target cells bearingADRP receptors and/or utilizing an ADRP lipid trafficking mechanism(e.g., pulmonary epithelial cells), cells while providing reducedbinding to non-target cells.

A) The Targeting Moiety (ADRP).

In various embodiments the targeting component/moiety comprising thechimeric moieties/structures of this invention is adipocytedifferentiation-related protein (ADRP) (see, e.g., GenBank Accession No:NP001113). The ADRP can be a full length ADRP or a fragment of ADRP ofsufficient length to specifically bind an ADRP receptor. The term“specifically binds”, as used herein, when referring to a protein orpolypeptide, refers to a binding reaction which is determinative of thepresence of the protein or polypeptide in a heterogeneous population ofproteins and other biologics. Thus, under designated conditions (e.g.immunoassay conditions in the case of an antibody), the specified ligand(e.g., ADRP) binds to its particular “target” (e.g. an ADRP receptor)and does not bind in a substantial amount to other proteins or receptorspresent in the sample or to other proteins to which the ADRP or chimericmoiety may come in contact in an organism.

A variety of assay formats can be used to identify ADRP fragments ofsufficient length to specifically bind to a ADRP receptor. Such assaysare performed in a manner analogous to the use of immunoassays todetermine specific binding of an antibody to a particular antigen.

Methods of screening a putative ligand for the ability to bind aparticular receptor are well known to those of skill in the art. Forexample, the receptor can be expressed or over expressed in a host cell.The cell can be contacted with a labeled ligand. After washing toeliminate free ligand, the cell can be screened for the presence of thelabeled ligand on the surface and/or internalized. Other screeningmethods are well known to those of skill in the art.

One of skill in the art will appreciate that analogues or fragments ofADRP bearing will also specifically bind to the ADRP receptor. Forexample, conservative substitutions of residues (e.g., a serine for analanine or an aspartic acid for a glutamic acid) comprising native ADRPwill provide ADRP analogues that also specifically bind to the ADRPreceptor. Thus, the term “ADRP”, when used in reference to a targetingmolecule, can also include fragments, analogues or peptide mimetics ofADRP that also specifically bind to the ADRP receptor. In preferredembodiments, these analogues and/or fragments and/or mimetics willparticipate in the ADRP lipid trafficking mechanism described herein.

In certain embodiments, to avoid degradation, e.g. if orally delivered,and/or to increase serum half-life, the targeting moiety, and/or theeffector can be protected with one or more blocking/protecting groups.Such groups include, but are not limited to polyethylene glycol (PEG),t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl group, anacetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a butyl, a pentyla hexyl ester, an N-methyl anthranilyl, and a 3 to 20 carbon alkyl,amide, a 3 to 20 carbon alkyl group, 9-fluoreneacetyl group,1-fluorenecarboxylic group, 9-fluorenecarboxylic group,9-fluorenone-1-carboxylic group, benzyloxycarbonyl (is also calledcarbobenzoxy mentioned above), Xanthyl (Xan), Trityl (Trt),4-methyltrityl (Mtt), 4-methoxytrityl (Mmt),4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl(Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBz1), 4-methoxybenzyl(MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom), cyclohexyloxy(cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu),trifluoroacetyl (TFA),4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}benzylester (ODmab), α-allyl ester (OAR), 2-phenylisopropyl ester (2-PhiPr),1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl (Dde), and the like.

In certain embodiments, to improve serum half-life, the targeting moietyand/or the effector can be circularly permuted. Circular permutation isfunctionally equivalent to taking a straight-chain molecule, fusing theends (directly or through a linker) to form a circular molecule, andthen cutting the circular molecule at a different location to form a newstraight chain molecule with different termini (see, e.g., Goldenberg,et al. J. Mol. Biol., 165: 407-413 (1983) and Pan et al. Gene 125:111-114 (1993)). Circular permutation thus has the effect of essentiallypreserving the sequence and identity of the amino acids of a proteinwhile generating new termini at different locations.

Circular permutation of ADRP provides a means by which the native ADRPprotein may be altered to produce new carboxyl and amino termini withoutdiminishing the specificity and binding affinity of the altered firstprotein relative to its native form. With new termini located away fromthe active (binding) site, it is possible to incorporate the circularlypermuted ADRP into a fusion protein with a reduced, or no diminution, ofADRP binding specificity and/or avidity.

It will be appreciated that while circular permutation is described interms of linking the two ends of a protein and then cutting thecircularized protein these steps are not actually required to create theend product. A protein can be synthesized de novo with the sequencecorresponding to a circular permutation of the native protein. Thus, theterm “circularly permuted ADRP (cpADRP)” refers to all ADRP proteinshaving a sequence corresponding to a circular permutation of ano-permuted (e.g., native) ADRP protein regardless of how they areconstructed.

Generally, however, a permutation that retains or improves the bindingspecificity and/or avidity (as compared to the native ADRP) ispreferred. If the new termini interrupt a critical region of the nativeprotein, binding specificity and avidity may be lost. Similarly, iflinking the original termini destroys ADRP binding specificity andavidity then no circular permutation is suitable. Thus, there aretypically two requirements for the creation of an active circularlypermuted protein: 1) The termini in the native protein are favorablylocated so that creation of a linkage does not destroy bindingspecificity and/or avidity; and 2) There exists an “opening site” wherenew termini can be formed without disrupting a region critical forprotein folding and desired binding activity (see, e.g., Thorton et al.(1983) J. Mol. Biol., 167: 443-460).

When circularly permuting ADRP, it is desirable to use a linker thatpreserves the spacing between the termini comparable to the unpermutedor native molecule. Generally linkers are either hetero- orhomo-bifunctional molecules that contain two reactive sites that mayeach form a covalent bond with the carboxyl and the amino terminal aminoacids respectively. Suitable linkers are well known to those of skill inthe art and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Themost common and simple example is a peptide linker that typicallyconsists of several amino acids joined through peptide bonds to thetermini of the native protein. The linkers can be joined to the terminalamino acids through their side groups (e.g., through a disulfide linkageto cysteine). However, in a preferred embodiment, the linkers are joinedto the alpha carbon amino and carboxyl groups of the terminal aminoacids.

Functional groups capable of forming covalent bonds with the amino andcarboxyl terminal amino acids are well known to those of skill in theart. For example, functional groups capable of binding the terminalamino group include anhydrides, carbodimides, acid chlorides, activatedesters and the like. Similarly, functional groups capable of formingcovalent linkages with the terminal carboxyl include amines, alcohols,and the like. In a preferred embodiment, the linker will itself be apeptide and will be joined to the protein termini by peptide bonds. Atypical linker for circular permutation and/or for joining components ofa fusion protein is Gly-Gly-Ser-Gly (SEQ ID NO:1)

One of skill in the art will appreciate that the ADRP can be modified ina variety of ways that do not destroy binding specificity and/or avidityand, in fact, may increase binding properties. Some modifications may bemade to facilitate the cloning, expression, or incorporation of the ADRPinto a fusion protein. Such modifications are well known to those ofskill in the art and include, for example, a methionine added at theamino terminus to provide an initiation site, or additional amino acidsplaced on either terminus to create conveniently located restrictionsites or termination codons.

One of skill will recognize that other modifications may be made. Thus,for example, amino acid substitutions may be made that increasespecificity or binding affinity of the circularly permuted protein, etc.Alternatively, non-essential regions of the molecule may be shortened oreliminated entirely. Thus, where there are regions of the molecule thatare not themselves involved in the activity of the molecule, they may beeliminated or replaced with shorter segments that merely serve tomaintain the correct spatial relationships between the active componentsof the molecule.

In certain embodiments, the chimeric moiety contains more than onetargeting molecule (e.g. a dual-targeted moiety). The chimeric moietycan contain, for example, targeting antibodies directed to tumor markersor other markers than the ADRP receptor. A number of such antibodies areknown and have even been converted to forms suitable for incorporationinto fusion proteins. These include anti-erbB2, B3, BR96, OVB3,anti-transferrin, Mik-B1 and PR1 (see Batra et al., Mol. Cell. Biol.,11: 2200-2205 (1991); Batra et al., Proc. Natl. Acad. Sci. USA, 89:5867-5871 (1992); Brinkmann, et al. Proc. Natl. Acad. Sci. USA, 88:8616-8620 (1991); Brinkmann et al., Proc. Natl. Acad. Sci. USA, 90:547-551 (1993); Chaudhary et al., Proc. Natl. Acad. Sci. USA, 87:1066-1070 (1990); Friedman et al., Cancer Res. 53: 334-339 (1993);Kreitman et al., J. Immunol., 149: 2810-2815 (1992); Nicholls et al., J.Biol. Chem., 268: 5302-5308 (1993); and Wells, et al., Cancer Res., 52:6310-6317 (1992), respectively).

B) The Effector.

As described above, the effector component of the chimeric structures ofthis invention can include any moiety whose activity it is desired todeliver to cells that express ADRP receptors and/or that participate inthe ADRP lipid trafficking mechanism described herein. Particularlypreferred effector molecules include therapeutic compositions such asliposomes and/or various drugs (e.g., retinoids) cytotoxins such as PEor DT, radionuclides, ligands such as growth factors, antibodies,detectable labels such as fluorescent or radioactive labels, and thelike.

1) Retinoic Acid, Analogues and Derivatives.

In certain embodiments, this invention contemplates the use of ADRPconstructs to specifically and/or preferentially deliver a retinoid to atarget tissue. Retinoids are useful in treating a wide variety ofepithelial cell carcinomas, including, but not limited to pulmonary,head, neck, esophagus, adrenal, prostate, ovary, testes, pancreas, andgut.

It is noted that ADRP is produced by the connective tissue cells thatunder lie alveolar cells and the ADRP receptor is found on the alveolarepithelium. The chimeric moieties of this invention are thusparticularly well suited to the specific and/or preferential delivery ofretinoids (and/or other moieties) to alveolar/pulmonary epithelium.

Retinoic acid, analogues, derivatives, and mimetics are well known tothose of skill in the art. Such retinoids include, but are not limitedto retinoic acid, ceramide-generating retinoid such as fenretinide (see,e.g., U.S. Pat. No. 6,352,844), 13-cis retinoic acid (see, e.g., U.S.Pat. Nos. 6,794,416, 6,339,107, 6,177,579. 6,124,485, etc.), 9-cisretinoic acid (see, e.g., U.S. Pat. Nos. 5,932,622, 5,929,057, etc.),9-cis retinoic acid esters and amides (see, e.g., U.S. Pat. No.5,837,728), 11-cis retinoic acid (see, e.g., U.S. Pat. No. 5,719,195),all trans retinoic acid (see, e.g., U.S. Pat. Nos. 4,885,311, 4,994,491,5,124,356, etc.), 9-(Z)-retinoic acid (see, e.g., U.S. Pat. Nos.5,504,230, 5,424,465, etc.), retinoic acid mimetic anlides (see, e.g.,U.S. Pat. No. 6,319,939), ethynylheteroaromatic-acids having retinoicacid-like activity (see, e.g., U.S. Pat. Nos. 4,980,484, 4,927,947,4,923,884 Ethynylheteroaromatic-acids having retinoic acid-likeactivity, 4,739,098, etc.) aromatic retinoic acid analogues (see, e.g.,U.S. Pat. No. 4,532,343), N-heterocyclic retinoic acid analogues (see,e.g., U.S. Pat. No. 4,526,7874), naphtenic and heterocyclic retinoicacid analogues (see, e.g., U.S. Pat. No. 518,609), open chain analoguesof retinoic acid (see, e.g., U.S. Pat. No. 4,490,414), entaerythritoland monobenzal acetals of retinoic acid esters (see, e.g., U.S. Pat. No.4,464,389), naphthenic and heterocyclic retinoic acid analogues (see,e.g., U.S. Pat. No. 4,456,618), azetidinone derivatives of retinoic acid(see, e.g., U.S. Pat. No. 4,456,618), and the like.

The retinoic acid, retinoic acid analogue, derivative, or mimetics canbe coupled (e.g., conjugated) to the targeting component (e.g. ADRP) orit can be contained within a liposome or complexed with a lipid that iscoupled to the targeting moiety, e.g. as described herein.

2) Other Cancer Therapeutics.

In certain embodiments the methods and compositions of this inventioncan be used to deliver other cancer therapeutics instead of or inaddition to the retinoic acid or retinoic acid analogue/derivative. Suchagents include, but are not limited to alkylating agents (e.g.,mechlorethamine (Mustargen), cyclophosphamide (Cytoxan, Neosar),ifosfamide (Ifex), phenylalanine mustard; melphalen (Alkeran),chlorambucol (Leukeran), uracil mustard, estramustine (Emcyt), thiotepa(Thioplex), busulfan (Myerlan), lomustine (CeeNU), carmustine (BiCNU,BCNU), streptozocin (Zanosar), dacarbazine (DTIC-Dome), cis-platinum,cisplatin (Platinol, Platinol AQ), carboplatin (Paraplatin), altretamine(Hexylen), etc.), antimetabolites (e.g. methotrexate (Amethopterin,Folex, Mexate, Rheumatrex), 5-fluoruracil (Adrucil, Efudex, Fluoroplex),floxuridine, 5-fluorodeoxyuridine (FUDR), capecitabine (Xeloda),fludarabine: (Fludara), cytosine arabinoside (Cytaribine, Cytosar,ARA-C), 6-mercaptopurine (Purinethol), 6-thioguanine (Thioguanine),gemcitabine (Gemzar), cladribine (Leustatin), deoxycoformycin;pentostatin (Nipent), etc.), antibiotics (e.g. doxorubicin (Adriamycin,Rubex, Doxil, Daunoxome-liposomal preparation), daunorubicin(Daunomycin, Cerubidine), idarubicin (Idamycin), valrubicin (Valstar),mitoxantrone (Novantrone), dactinomycin (Actinomycin D, Cosmegen),mithramycin, plicamycin (Mithracin), mitomycin C (Mutamycin), bleomycin(Blenoxane), procarbazine (Matulane), etc.), mitotic inhibitors (e.g.paclitaxel (Taxol), docetaxel (Taxotere), vinblatine sulfate (Velban,Velsar, VLB), vincristine sulfate (Oncovin, Vincasar PFS, Vincrex),vinorelbine sulfate (Navelbine), etc.), chromatin function inhibitors(e.g., topotecan (Camptosar), irinotecan (Hycamtin), etoposide (VP-16,VePesid, Toposar), teniposide (VM-26, Vumon), etc.), hormones andhormone inhibitors (e.g. diethylstilbesterol (Stilbesterol,Stilphostrol), estradiol, estrogen, esterified estrogens (Estratab,Menest), estramustine (Emcyt), tamoxifen (Nolvadex), toremifene(Fareston) anastrozole (Arimidex), letrozole (Femara),17-OH-progesterone, medroxyprogesterone, megestrol acetate (Megace),goserelin (Zoladex), leuprolide (Leupron), testosteraone,methyltestosterone, fluoxmesterone (Android-F, Halotestin), flutamide(Eulexin), bicalutamide (Casodex), nilutamide (Nilandron), etc.)INHIBITORS OF SYNTHESIS (e.g., aminoglutethimide (Cytadren),ketoconazole (Nizoral), etc.), immunomodulators (e.g., rituximab(Rituxan), trastuzumab (Herceptin), denileukin diftitox (Ontak),levamisole (Ergamisol), bacillus Calmette-Guerin, BCG (TheraCys, TICEBCG), interferon alpha-2a, alpha 2b (Roferon-A, Intron A),interleukin-2, aldesleukin (ProLeukin), etc.) and other agents such as1-asparaginase (Elspar, Kidrolase), pegaspargase (Oncaspar), hydroxyurea(Hydrea, Doxia), leucovorin (Wellcovorin), mitotane (Lysodren), porfimer(Photofrin), tretinoin (Veasnoid), and the like.

3) Cytotoxins.

In certain embodiments, the effector comprises a cytotoxin (e.g. to killa tumor cell). Suitable cytotoxins include, but are not limited toPseudomonas exotoxins, Diphtheria toxins, ricin, abrin, and the like.

Pseudomonas exotoxin A (PE) is an extremely active monomeric protein(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, whichinhibits protein synthesis in eukaryotic cells through the inactivationof elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation(catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD ontoEF-2).

The toxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2, which inactivates the protein andcauses cell death. The function of domain Ib (amino acids 365-399)remains undefined, although a large part of it, amino acids 365-380, canbe deleted without loss of cytotoxicity. See Siegall et al., J. Biol.Chem. 264: 14256-14261 (1989).

Where the targeting moiety (e.g. ADRP) is fused to PE, one preferred PEmolecule is one in which domain Ia (amino acids 1 through 252) isdeleted and amino acids 365 to 380 have been deleted from domain Ib.However all of domain Ib and a portion of domain II (amino acids 350 to394) can be deleted, particularly if the deleted sequences are replacedwith a linking peptide such as GGGGS (SEQ ID NO:2).

In addition, the PE molecules can be further modified usingsite-directed mutagenesis or other techniques known in the art, to alterthe molecule for a particular desired application. Means to alter the PEmolecule in a manner that does not substantially affect the functionaladvantages provided by the PE molecules described here can also be usedand such resulting molecules are intended to be covered herein. Suchmodified PE molecules are known to those of skill in the art andinclude, but are not limited to the incorporation of one or moretranslocation sequences (e.g., REDL, RDEL, KDEL, etc.) (see, e.g.,Chaudhary et al. (1991) Proc. Natl. Acad. Sci. USA 87:308-312 andSeetharam et al. (1991) J. Biol. Chem. 266: 17376-173810, deletions ofamino acids 365-380 of domain Ib, substitution of methionine at aminoacid position 280 in place of glycine, and the like (see, e.g., Debinskiet al. (1994) Bioconj. Chem., 5: 40).

Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 thereby inhibiting protein synthesis. Diphtheriatoxin, however, is divided into two chains, A and B, linked by adisulfide bridge. In contrast to PE, chain B of DT, which is on thecarboxyl end, is responsible for receptor binding and chain A, which ispresent on the amino end, contains the enzymatic activity (Uchida et al.(1972) Science, 175: 901-903; Uchida et al. (1973) J. Biol. Chem., 248:3838-3844).

In certain embodiments, the targeting moiety-Diphtheria toxin fusionproteins of this invention have the native receptor-binding domainremoved by truncation of the Diphtheria toxin B chain. Particularlypreferred is DT388, a DT in which the carboxyl terminal sequencebeginning at residue 389 is removed (see, e.g., Chaudhary, et al. (1991)Bioch. Biophys. Res. Comm., 180: 545-551).

Like the PE chimeric cytotoxins, the DT molecules can be chemicallyconjugated to the ADRP targeting moiety, but, in a preferred embodiment,the targeting moiety is fused to the Diphtheria toxin by recombinantmeans. The genes encoding protein chains may be cloned in cDNA or ingenomic form by any cloning procedure known to those skilled in the art.Methods of cloning genes encoding DT fused to various ligands are alsowell known to those of skill in the art (see, e.g., Williams et al.(1990) J. Biol. Chem. 265: 11885-11889).

The term “Diphtheria toxin” (DT) as used herein refers to full lengthnative DT or to a DT that has been modified. Modifications typicallyinclude removal of the targeting domain in the B chain and, morespecifically, involve truncations of the carboxyl region of the B chain.

4) Detectable Labels.

Detectable labels suitable for use as the effector molecule component ofthe chimeric molecules of this invention include any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include magnetic beads (e.g. DYNABEADS™), fluorescent dyes(e.g., fluorescein isothiocyanate, texas red, rhodamine, greenfluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S,¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.polystyrene, polypropylene, latex, etc.) beads.

It will be recognized that labels are not to be limited to singlespecies organic molecules, but include inorganic molecules,multi-molecular mixtures of organic and/or inorganic molecules,crystals, heteropolymers, and the like. Thus, for example, CdSe—CdScore-shell nanocrystals enclosed in a silica shell can be easilyderivatized for coupling to a biological molecule (Bruchez et al. (1998)Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots(zinc sulfide-capped cadmium selenide) have been covalently coupled tobiomolecules for use in ultrasensitive biological detection (Warren andNie (1998) Science, 281: 2016-2018).

Means of detecting labels are well known to those of skill in the art.Thus, for example, radiolabels may be detected using photographic filmor scintillation counters, fluorescent markers may be detected using aphotodetector to detect emitted illumination. Enzymatic labels aretypically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

5) Ligands.

As explained above, the effector molecule may also comprise a ligand oran antibody. In certain embodiments, the ligands and antibodies arethose that bind to surface markers on immune cells. Chimeric moleculesutilizing such antibodies as effector molecules act as bifunctionallinkers establishing an association between the immune cells bearingbinding partner(s) for the ligand or antibody and the target cellsexpressing the ADRP receptor. Suitable antibodies and growth factors areknown to those of skill in the art and include, but are not limited to,IL-2, IL-4, IL-6, IL-7, tumor necrosis factor (TNF), anti-Tac, TGFα, andthe like.

6) Other Therapeutic Moieties.

Other suitable effector molecules include various pharmacological agentsand/or encapsulation systems containing various pharmacological agents.Thus, the targeting molecule of the chimeric molecule can be attacheddirectly to a drug (e.g. a drug that is to be delivered directly to atumor). Such drugs are well known to those of skill in the art andinclude, but are not limited to, doxirubicin, vinblastine, genistein,taxol, antisense molecules, and the like.

In certain embodiments, the effector molecule can comprise anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (e.g. anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735,Connor et al., Pharm. Ther., 28: 341-365 (1985)

C. Attachment of the Targeting Moiety to the Effector.

One of skill will appreciate that the targeting moiety (e.g., ADRP) andthe effector(s) can be joined together in any order. Thus, for example,the effector can be joined to either the amino or carboxy terminal ofthe ADRP. The ADRP can may also be joined to an internal region of theeffector, or conversely, the effector can be joined to an internallocation of the ADRP, as long as the attachment does not interfere withthe respective activities of the components.

The targeting moiety and the effector can be attached by any of a numberof means well known to those of skill in the art. In certainembodiments, the effector is conjugated, either directly or through alinker (spacer), to the targeting moiety. Where both the effector andthe targeting moiety are polypeptides, however, it can be preferable torecombinantly express the chimeric moiety as a fusion protein.

a) Conjugation of the Effector Molecule to the Targeting Molecule.

In certain embodiments, the targeting moiety (e.g., ADP, cpADRP, oranti-ADRPR antibody) is chemically conjugated to the effector molecule(e.g., a liposome, a retinoic acid, a cytotoxin, a label, a ligand,etc.). Means of chemically conjugating molecules are well known to thoseof skill.

The procedure for attaching an agent to polypeptide or other targetingmoiety will vary according to the chemical structure of the agent.Polypeptides typically contain variety of functional groups; e.g.,carboxylic acid (COOH) or free amine (—NH₂) groups, which are availablefor reaction with a suitable functional group on an effector molecule tobind the effector thereto.

Alternatively, the targeting molecule and/or effector molecule can bederivatized to expose or attach additional reactive functional groups.The derivatization can involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, typically refers to a molecule that is usedto join the targeting moiety to the effector. In various embodiments,the linker is capable of forming covalent bonds to both the targetingmoiety and to the effector. Suitable linkers are well known to those ofskill in the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the targeting moiety and the effector are polypeptides,the linker(s) can be joined to the constituent amino acids through theirside groups (e.g., through a disulfide linkage to cysteine). However, ina certain preferred embodiments, the linkers are be joined to the alphacarbon amino and/or carboxyl groups of the terminal amino acids.

A bifunctional linker having one functional group reactive with a groupon a particular agent, and another group reactive with a protein (e.g.,ADRP), may be used to form the desired conjugate. Alternatively,derivatization can involve chemical treatment of the targeting moiety.Procedures for generation of, for example, free sulfhydryl groups onpolypeptides, such as antibodies or antibody fragments, are known (SeeU.S. Pat. No. 4,659,839).

Many procedures and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins suchas antibodies are known. See, for example, European Patent ApplicationNo. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987)Cancer Res. 47: 4071-4075. In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,”Thorpe et al., Monoclonal Antibodies in Clinical Medicine, AcademicPress, pp. 168-190 (1982); Waldmann (1991) Science, 252: 1657; U.S. Pat.Nos. 4,545,985 and 4,894,443, and the like.

In some circumstances, it is desirable to free the effector from thetargeting molecule when the chimeric molecule has reached its targetsite. Therefore, chimeric conjugates comprising linkages which arecleavable in the vicinity of the target site can be used when theeffector is to be released at the target site. Cleaving of the linkageto release the agent from the targeting moiety can be prompted byenzymatic activity or conditions to which the conjugate is subjectedeither inside the target cell or in the vicinity of the target site.When the target site is a tumor, a linker which is cleavable underconditions present at the tumor site (e.g. when exposed totumor-associated enzymes or acidic pH) may be used.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers which are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient's complementsystem. In view of the large number of methods that have been reportedfor attaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

b) Production of Fusion Proteins.

Where the targeting moiety (e.g., ADRP) and/or the effector isrelatively short (i.e., less than about 50 amino acids) they may besynthesized using standard chemical peptide synthesis techniques. Whereboth molecules are relatively short the chimeric moiety can besynthesized as a single contiguous polypeptide. Alternatively thetargeting moiety and the effector can be synthesized separately and thenfused by condensation of the amino terminus of one molecule with thecarboxyl terminus of the other molecule thereby forming a peptide bond.Alternatively, the targeting moiety and the effector can each becondensed with one end of a peptide spacer molecule thereby forming acontiguous fusion protein.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield (1963)Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156; and Stewart etal. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill.

In certain embodiments, chimeric fusion proteins of the presentinvention are synthesized using recombinant DNA methodology. Generallythis involves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion proteins (e.g. ADRP-effector) of this inventioncan be prepared by any suitable method, including, for example, cloningand restriction of appropriate sequences or direct chemical synthesis bymethods such as the phosphotriester method of Narang et al. (1979) Meth.Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979)Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method ofBeaucage et al. (1981) Tetra. Lett., 22: 1859-1862); the solid supportmethod of U.S. Pat. No. 4,458,066, and the like.

Chemical synthesis produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences can be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments can then be ligated to produce the desired DNA sequence.

In certain embodiments, DNA encoding fusion proteins of the presentinvention can be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, for example, the gene for ADRP isPCR amplified, using a sense primer containing the restriction site forNdeI and an antisense primer containing the restriction site forHindIII. This can produce a nucleic acid encoding the mature ADRPsequence and having terminal restriction sites. An effector having“complementary” restriction sites can similarly be cloned and thenligated to the ADRP targeting moiety and/or to a linker attached to theADRP targeting moiety. Ligation of the nucleic acid sequences andinsertion into a vector produces a vector encoding ADRP joined to theeffector.

While the two molecules can be directly joined together, one of skillwill appreciate that the molecules can be separated by a peptide spacerconsisting of one or more amino acids. Generally the spacer will have nospecific biological activity other than to join the proteins or topreserve some minimum distance or other spatial relationship betweenthem. However, the constituent amino acids of the spacer may be selectedto influence some property of the molecule such as the folding, netcharge, or hydrophobicity.

The nucleic acid sequences encoding the fusion proteins can be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene istypically operably linked to appropriate expression control sequencesfor each host. For E. coli this includes a promoter such as the T7, trp,or lambda promoters, a ribosome binding site and preferably atranscription termination signal. For eukaryotic cells, the controlsequences will include a promoter and preferably an enhancer derivedfrom immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes (1982) ProteinPurification, Springer-Verlag, N.Y.: Deutscher (1990) Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y., and the like).

Substantially pure compositions of at least about 90 to 95% homogeneityare preferred, and 98 to 99% or more homogeneity are most preferred forpharmaceutical uses. Once purified, partially or to homogeneity asdesired, the polypeptides may then be used therapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the ADRP targeted fusion proteinmay possess a conformation substantially different than the nativeconformations of the constituent polypeptides. In this case, it may benecessary to denature and reduce the polypeptide and then to cause thepolypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing proteins and inducing re-folding are well knownto those of skill in the art (see, e.g., Debinski et al. (1993) J. Biol.Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4:581-585; and Buchner, et al. (1992) Anal. Biochem., 205: 263-270).Debinski et al., for example, describe the denaturation and reduction ofinclusion body proteins in guanidine-DTE. The protein is then refoldedin a redox buffer containing oxidized glutathione and L-arginine.

One of skill would recognize that modifications can be made to theADRP-fusion proteins without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons.

III. Identification/Validation of Target Cells.

It was a surprising discovery of the present invention that ADRP and/orADRP-chimeric moieties bind to the epithelial cell surface and aretransported to the nucleus. Since the targeting mechanism directs ADRPfrom the circulation to the lung epithelium, it can be exploited todeliver therapeutic retinoic acid derivatives or other moieties directlyto the epithelium, e.g. to image or to treat lung cancer.

Thus, the methods of this invention can be used to target an effectormolecule to a variety of cells including, but not limited to a number ofepithelial cells (e.g., pulmonary, head, neck, esophagus, adrenal,prostate, ovary, testes, pancreas, gut, and the like). Neoplasias ofthese tissues are well known to those of skill in the art and include,but are not limited to, cancers of the reproductive system (e.g.,testicular, ovarian, cervical), cancers of the gastrointestinal tract(e.g., stomach, small intestine, large intestine, colorectal, etc.),cancers of the head and neck, lung cancers, prostate cancers (e.g.,prostate carcinoma), kidney cancers, lung cancers (e.g., mesothelioma),pancreatic cancers, and the like.

One of skill in the art will appreciate that identification andconfirmation of effective targeting of a cell or tissue by theconstructs of this invention requires only routine screening usingwell-known methods. Typically this involves providing, e.g. a labeledADRP (e.g. a radioactive or GFP labeled ADRP). Specific/preferential invitro and/or in vivo targeting using such a moiety can readily beevaluated, e.g., as described in Example 1.

IV. Pharmaceutical Compositions.

The chimeric moieties of this invention can be useful for parenteral,topical, oral, or local administration, such as by aerosol ortransdermally, for prophylactic and/or therapeutic treatment. Thechimeric moieties can be formulated into pharmacological compositions(e.g., combination with an appropriate excipient). The pharmacologicalcompositions can be administered in a variety of unit dosage formsdepending upon the method of administration. For example, unit dosageforms suitable for oral administration include powder, tablets, pills,capsules and lozenges. It is recognized that the chimeric moieties andpharmaceutical compositions thereof, when administered orally, aretypically protected from digestion. This is typically accomplishedeither by complexing the active component(s) with a composition torender it resistant to acidic and enzymatic hydrolysis or by packagingthe composition in an appropriately resistant carrier such as aliposome. Means of protecting proteins and other compositions fromdigestion are well known in the art.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. Thecompositions for administration will commonly comprise a solution of thechimeric moiety dissolved or suspended in a pharmaceutically acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., buffered saline and the like. These solutions aresterile and generally free of undesirable matter. These compositions canbe sterilized by conventional, well known sterilization techniques. Thecompositions can contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofchimeric moiety in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., (1980) Mack Publishing Company,Easton, Pa.

The compositions containing the chimeric moieties and/or a combinationof other active agents can be administered for therapeutic treatments.In therapeutic applications, compositions are administered to a patientsuffering from a disease, in an amount sufficient to cure or at leastpartially arrest the disease and its complications. An amount adequateto accomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the proteins of this invention to effectivelytreat the patient.

Among various uses of the chimeric moieties described herein is themitigation or elimination of one or more symptoms of a cancer (e.g., acancer of an epithelial tissue such as a pulmonary cancer). Onepreferred application is the treatment of cancer, such as by the use ofan ADRP coupled to a retinoic acid or derivative thereof.

It will be appreciated by one of skill in the art that there are someregions that are not heavily vascularized or that are protected by cellsjoined by tight junctions and/or active transport mechanisms whichreduce or prevent the entry of macromolecules present in the bloodstream. Thus, for example, systemic administration of therapeutics totreat gliomas, or other brain cancers, is constrained by the blood-brainbarrier which resists the entry of macromolecules into the subarachnoidspace.

One of skill in the art will appreciate that in these instances, thetherapeutic compositions of this invention can be administered directlyto the tumor site. Thus, for example, certain tumors can be treated byadministering the therapeutic composition directly to the tumor site(e.g., through a surgically implanted catheter). Where the fluiddelivery through the catheter is pressurized, small molecules (e.g. thetherapeutic molecules of this invention) will typically infiltrate asmuch as two to three centimeters beyond the tumor margin.

Alternatively, the therapeutic composition can be placed at the targetsite in a slow release formulation. Such formulations can include, forexample, a biocompatible sponge or other inert or resorbable matrixmaterial impregnated with the therapeutic composition, slow dissolvingtime release capsules or microcapsules, and the like.

Typically the catheter or time release formulation will be placed at thetumor site as part of a surgical procedure. Thus, for example, wheremajor tumor mass is surgically removed, the perfusing catheter or timerelease formulation can be emplaced at the tumor site as an adjuncttherapy. Of course, surgical removal of the tumor mass may be undesired,not required, or impossible, in which case, the delivery of thetherapeutic compositions of this invention may comprise the primarytherapeutic modality.

VII. Kits.

In certain embodiments, this invention provides for kits for thetreatment of tumors or for the detection of certain cells (e.g. cellsexpressing ADRP receptor s and/or participating in the ADRP lipidtrafficking mechanism described herein). Kits typically comprise achimeric moiety of the present invention (e.g. ADRP-label,ADRP-liposome/retinoic acid, ADRP-ligand, etc.). In addition the kitstypically include instructional materials disclosing means of use of thechimeric moiety (e.g. as a therapeutic for a pulmonary cancer, fordetection of tumor cells, to augment an immune response, etc.). The kitsmay also include additional components to facilitate the particularapplication for which the kit is designed. Thus, for example, where akit contains a chimeric molecule in which the effector molecule is adetectable label, the kit can additionally contain means of detectingthe label (e.g. enzyme substrates for enzymatic labels, filter sets todetect fluorescent labels, appropriate secondary labels such as a sheepanti-mouse-HRP, or the like). The kits can additionally include buffersand other reagents routinely used for the practice of a particularmethod. Such kits and appropriate contents are well known to those ofskill in the art.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 ADRP Coordinates Surfactant Phospholipid and ProteinExpression

Materials and methods.

Materials:

A549 cells were obtained from the ATCC, Rockville, Md. Streptomycin,penicillin and RPMI 1640 medium were obtained from Life Technologies(Gaithersburg, Md.). Fetal Bovine Serum was purchased from Hyclone(Logan, Utah). Radiolabeled ³H-triolein was purchased from New EnglandNuclear, Boston, Mass. Time-mated Sprague Dawley rats were purchasedfrom Charles River Breeders, (Holister, Calif.). Animals were treated inaccordance with NIH Guidelines, and the protocol was approved by the LosAngeles Biomedical Research Institute. Antibody against SurfactantProtein-B (SP-B) was purchased from Santa Cruz. Biotechnology, Inc(Santa Cruz, Calif.).

Immunoblotting:

Whole lung tissue from pre- and postnatal rats was excised, rinsed inPBS, snap-frozen in liquid N₂, and stored at −80° C. until furtherprocessing. Frozen lung tissue was homogenized with a Teflon homogenizerin ice-cold hypotonic lysis medium containing 10 mM Tris HCl, pH 7.4, 1mM EDTA, 10 mM sodium fluoride, 20 μg/ml leupeptin, 1 mM benzamidine,and 100 μM [4-(2-aminoethyl)-benzenesulfonylfluoride]hydrochloride.Protein concentration was measured with a dye binding assay (BioRad) asper the manufacturer's protocol. Aliquots of homogenates (100 μg) wereelectrophoresed under denaturing SDS-PAGE conditions according toLaemmli (Laemmli (1970) Nature 227(259): 680-685) in 10% gels.Immunoblotting was performed by the method of Towbin et al. (Towbin etal. (1979) Proc Natl Acad Sci USA. 76(9): 4350-4354). Blots wereincubated for 1 h at 25° C. in blocking solution (composed of 5% milk inTBS (10 mM Tris, 0.15 M NaCl, and 0.5% Tween 20 at pH 7.4)) andincubated at 25° C. with a monoclonal antibody (IgG in culture mediumdiluted 1:10 in blocking solution) against an epitope within the first25 amino acids of adipophilin, human ADRP (Research Diagnostics). After2 h, the blots were washed five times (10 min each) in TBS, incubatedfor 1 h with alkaline-phosphatase-conjugated goat anti-mouse IgG[Jackson ImmunoResearch (1:2,000 in blocking solution)], and finallywashed five times (10 min each) with TBS. ADRP protein was detected byreaction of immuno-bound alkaline-phosphatase with5-bromo-4-chloro-3-indoylphosphate p-toluidine and p-nitro bluetetrazolium chloride as per the manufacturer's instructions (BioRad).

Culture of Fetal Rat Lung Fibroblasts:

Fetal rat lung fibroblasts were prepared and cultured as previouslydescribed (Floros et al. (1987) J Biol. Chem. 262(28): 13592-13598;Torday et al. (2001) Pediatr Res. 49(6): 843-849).

Culture of A549 Cells:

A549 cells were propagated in monolayer in RPMI medium containing 10%fetal bovine serum at 37° C. in an atmosphere of 5% CO₂/air.

Preparation of ³H-Triglyceride-Labeled Lipid Droplets.

Fetal lung fibroblast monolayers were incubated with ³H-triolein (5:Ci/ml) for 12 h (Torday et al. (1995) Biochim Biophys Acta.,1254(2):198-206). The cells were gently scraped into PBS and pelleted bycentrifugation at 500×g for 5 min at room temperature. ADRP-coated lipiddroplets were then isolated from the fibroblasts by differentialcentrifugation as follows: all pipettes, homogenizers, and tubes weresiliconized. Cells were disrupted by incubation in hypotonic lysismedium (as described under Immunoblotting) containing 10% glycerol for15 min at room temperature followed by 15 strokes in a Teflon-glasshomogenizer. The homogenate was centrifuged at 500×g for 5 min. Theresulting supernatant (containing lipid droplets) was adjusted to 5-10%sucrose and centrifuged at 50,000×g for 30 min at 4° C., resulting inthe lipid droplets forming a floating cake at the top of the centrifugetube. The lipid cake was removed, resuspended, and homogenized in freshlysis medium containing glycerol, and again centrifuged at 50,000×g for30 min at 4° C. The amounts of non-radioactive and radiolabeledtriglycerides in the lipid droplet fraction were determined byextraction, chromatography, and quantitation of the triglyceridefraction as previously described (Schultz et al. (2002) Am. J. Physiol.Lung Cell Mol. Physiol. 283(2): L288-1296). These washed, ³H-labeledlipid droplets were then incubated with cultured A549 cells as describedbelow.

GFP-ADRP Fusion Constructs Expressed in CHO Fibroblasts:

ADRP gene-specific primers were designed from the human ADRP (hADRP)mRNA sequence (GenBank™ accession number BC005127). In conjunction withthe Access RT-PCR System (Promega), these primers generate hADRP cDNAfrom total RNA prepared from the human hepatoma cell line, HuH-7. TotalRNA is made using Trizol reagent (Sigma) according to the manufacturer'sinstructions. The hADRP cDNA is ligated to pGEM-T Easy (Promega) and,from the resultant progeny after transformation, plasmids are screenedfor the presence of hADRP sequences by restriction endonucleasedigestion. Plasmid DNA from positive clones was isolated and thenucleotide sequence of the hADRP cDNA was determined. For the mouse ADRP(mADRP) cDNA, an amplified product was generated using mRNA isolatedfrom mouse L cells and primers (CTA TGG CAG CAG CAG TAG TGG ATC CG (SEQID NO:3) and TCA TCT GGC CAG CAA CAT CAT GCT (SEQ ID NO:4)) that werederived from murine sequences generated in the Londos laboratory, NIDDK(GenBank accession number NM007408). The PCR product was inserted intopCRII-TOPO (In vitrogen). A clone containing an mADRP cDNA insert,pCRII/mADRP, was selected by restriction enzyme analysis and thenucleotide sequence of the inserted fragment was determined.

Construction of Plasmids Expressing DNase X, Human and Mouse ADRP Fusedto Fluorescent Proteins:

The hADRP ORF is excised from pLA1 using EcoRI and ligated to EcoRIlinearized pEGFPC1 (Clontech) to generate a plasmid termed pLA4 thatencodes a GFP-hADRP fusion protein. The same strategy is employed tofuse the hADRP ORF to YFP in plasmid pEYFP-C1 (Clontech). Plasmid pLA5,encoding the N-terminal half of hADRP linked to the C terminus of GFP,was constructed by digesting pLA4 with BamHI, which removed the 3′terminal region of the hADRP ORF, followed by re-circularization of thedigested plasmid. To fuse the C-terminal region of hADRP to GFP, thesmaller DNA fragment liberated upon digestion of pLA4 with BamHI ispurified and ligated to BamHI linearized pEGFP-C1. This construct isreferred to as pLA14. Constructs pLA10, pLA9, and pLA13 encode GFP-hADRPfusion proteins in which premature stop codons are inserted into thecoding sequence by site-directed mutagenesis using the Altered Sites IImammalian in vitro mutagenesis system (Promega). This system requiresthe use of oligonucleotides containing single nucleotide mismatchescorresponding to the region of the hADRP ORF to be mutated.Oligonucleotides TTC TAG TTC TTA CTC AGT GAG (SEQ ID NO:5), GCT CAC GAGCTA CAT CAT CCG (SEQ ID NO:6) and CCC TTT GGT CTA GTC CAT CAC (SEQ IDNO:7) are used to generate the GFP-hADRP fusion proteins encoded bypLA10, pLA9, and pLA13, respectively, and premature stop codons areinserted at nucleotide positions 671-673 (pLA10), 593-595 (pLA9), and503-505 (pLA13) within the hADRP ORF (nucleotides numbered according tothe ADRP sequence in BC005 127). N-terminal deletions of the hADRP ORFare created by using oligonucleotides TGT GAG ATG GCA GAG AAC GGT (SEQID NO:8) and GAC CTC ATG TCC TCA GCC TAT (SEQ ID NO:9) to amplifyregions of the ADRP ORF from internal ATG codons situated at nucleotides230-232 (pLA1 1) and 170-172 (pLA12), respectively. In both PCRreactions, the downstream primer used is AGA CAG GGA TCC CAG TCT AAC(SEQ ID NO:10), which terminates amplification of sequences after theBamHI site is located within the hADRP ORF. The resulting hADRP DNAfragments were ligated into pGEM-T Easy. EcoRI digestion is used toliberate the hADRP DNA fragments from pGEM-T Easy, which are theninserted into EcoRI-linearized pEGFP-C1. To generate pLA17, ligation isperformed with hADRP DNA fragments that are liberated upon digestion ofpLA12 with EcoRI and BamHI and pLA4 with BamHI and SalI together withpEGFP-C1 linearized with EcoRI and SalI. pLA22 was made by digestingpLA4 with MscI, which removed the region of the hADRP ORF betweennucleotides 267 and 476 (inclusive), followed by purification andre-circularization of the digested plasmid using T4 DNA ligase.Construct pLA29 was created by digesting pLA4 with MscI and BamHI, whichremoves the region of the hADRP ORF between nucleotides 267 and 746(inclusive). The digested plasmid was purified, treated with Klenowenzyme, and re-circularized using T4 DNA ligase.

For expression of mADRP, a BamHI/SpeI fragment from pCRII/mADRPcontaining mADRP nucleotide sequences was inserted first into pGEM-1(Promega) cleaved with HindIII/XbaI along with the oligonucleotide mADRPsequence AGC TTG GAT CCA TGG CAG CAG CAG TAG TA (SEQ ID NO:11). Becausethe BamHI/SpeI fragment removes part of the mADRP coding region (theBamHI site lies 15 nucleotides downstream of the ATG initiation codon),oligonucleotide mADRP1 restores these sequences. Inserting thisoligonucleotide also abolishes the BamHI site in the mADRP coding regionwithout altering the predicted amino acid sequence and places a novelBamHI site immediately upstream of the ATG codon. The resulting clone istermed pGEM/mADRP. A BamHI fragment from pGEM/mADRP that is introducedinto pEGFP-C1 also cleaves with BamHI to give plasmid pGFP-mADRP.

The vector pGFP-DNase X, which directs the synthesis of a GFP-DNase Xfusion product, was obtained by initially subcloning a PCR-generatedcDNA fragment containing the complete coding region of DNase X into themammalian expression vector pcDNA3.1 (Invitrogen). The DNase X codingregion is fused N-terminal to GFP in pEGFP (Clontech), to givepGFP-DNase X.

Maintenance of Tissue Culture Cells and Generation of Cells ExpressingGFP-mADRP

HuH-7 and Vero cells were propagated in Dulbecco's modified Eagle'smedium supplemented with 10% fetal calf serum, 2 mM L-glutamine,non-essential amino acids, and 100 IU/ml penicillin/streptomycin. Togenerate cells constitutively expressing GFP-mADRP, CHO cells weretransfected with plasmid pGFP-mADRP followed by selection with 800 μg/mlG418 (Clontech). Clones producing GFP-mADRP were selected first bypooling GFP fluorescent cells isolated by fluorescent-activated cellsorting (Beckman) followed by growth of individual colonies. Cloned celllines were maintained in media containing 500 μg/ml G418.

Preparation of GFP-Labeled Lipid Droplets.

CHO fibroblasts expressing GFP fused to ADRP were incubated with 400 μMoleic acid [coupled to fatty acid-free bovine serum albumin (BSA) at aratio of 6:1 mol/mol] 24 h at 37° C. in an atmosphere of 5% CO₂-air.(Brasaemle et al. (2000) J Biol. Chem. 275(49): 38486-38493). Lipiddroplets were processed as in the case of the ³H-triglyceride-labeledlipid droplets (see above). The amount of triglycerides in the lipiddroplet fraction was determined by extraction, chromatography, andquantitation of the triglyceride (Torday et al. (1995) Biochim BiophysActa., 1254(2):198-206).

Incubation of A549 Cells with Lipid Droplets:

Monolayer cultures of A549 cells were incubated with ADRP-LDs labeledwith either tritium or GFP as follows: ³H-labeled (10,000 dpm/min/20 μgtriacylglycerol) ADRP-LDs were isolated from fetal rat lung fibroblasts,as described above. Where indicated, incubations were carried out in thepresence of either actinomycin D or cyclohexamide to determine if denovo mRNA or protein synthesis, respectively, was involved in LDprocessing. Elsewhere, incubations were conducted in the presence ofADRP antibody (rabbit anti-ADRP IgG kindly provided by Dr. ConstantineLondos, NIDDK) to determine the specificity of the ADRP effect on LDuptake. At the end of the incubation, the cells were processed for³H-satPC (Floros et al. (1987) J Biol. Chem. 262(28): 13592-13598),expression of SP-B by RT-PCR and Western Blot (Rehan et al. (2002) MolGenet Metab. 76(1): 46-56), or for confocal microscopy (see below formethod).

Determination of Surfactant Phospholipid Synthesis:

The rates of satPC synthesis were determined as previously described(Floros et al. (1987) J Biol. Chem. 262(28): 13592-13598).

Determination of SP-B Expression:

Western blot and RT-PCR for SP-B were performed as described by Rehan etal. (2002) Mol Genet Metab. 76(1): 46-56.

Confocal Microscopy for Documentation of Nuclear Localization:

A549 cells cultured on circular, 1-mm-thick, glass coverslips (Red LabelMicro Cover Glasses; Thomas Scientific) were washed three times withPBS, fixed in 3% paraformaldehyde at pH 7.4 for 60 minutes, washed againthree times with PBS, and stored in fresh PBS (0-12 hours) at 4° C.until immunostained. The cells were permeabilized with 1% saponin, whichwas present in all incubations after fixation. Fixed cells were washed,incubated for 60 min in quenching/blocking solution (PBS containing 0.2M glycine and 1.25 mg of goat IgG/ml), incubated for 18 hours at 4° C.with antibody against ADRP, washed three times with PBS (10 minuteseach), incubated for 60 minutes with labeled secondary antibody, andwashed again three times with PBS (10 minutes each). Cells wereinverted, mounted on coverslips and viewed on a Leica TCS SP II confocalmicroscope with appropriate filters for fluorescein or rhodamine.

Intravenous Injection of GFP-ADRP Lipid Droplets:

Adult Sprague-Dawley rats were injected intravenously with GFP-ADRP LDs(x, y, z micrograms triglyceride equivalent) and sacrificed with anoverdose of pentobarbital 30 minutes post-injection. The lungs wereextirpated, perfused x-times with cold PBS to purge them of vascularGFP-ADRP LDs. The lung tissue (rt upper lobe) was snap frozen in liquidnitrogen and kept at −80° C. until further analysis. Lung tissue wasprocessed for GFP using Western Blot technique as described by Rehan etal. (2002) Mol Genet Metab. 76(1): 46-56.

Statistical Analyses

Data were analyzed by Analysis of Variance with the Student-Newman-Keulspost-hoc test and t-test as appropriate (Id.).

Results.

Nuclear Translocation of GFP-ADRP in Culture.

Time-Course for A549 Uptake and Localization of GFP-ADRP Complexes:

Upon incubation of A549 cells with GFP-ADRP LDs for 10 minutes (FIG. 1),there was rapid uptake and transit of the complex to the perinuclearregion of these cells. After 2 hours the GFP complexes appeared asprominent inclusions in the cytoplasm and perinuclear region of thesecells, becoming more diffusely spread over the entire cell by 24 hours.

Dose-Dependent A549 Uptake and Localization of GFP-ADRP Complexes:

A549 cells were incubated for 2 h with 0 to 100 :g/ml triglycerideequivalents of GFP-ADRP LD (FIG. 2). There were a few cells containingLDs at the 50 :g/ml dose; at 100 :g/ml there was prominent localizationof LDs in the perinuclear (see arrows) and cytoplasmic regions of thesecells.

Uptake of ADRP by A549 Cells Induces SP-B Expression:

A549 cell monolayer cultures were incubated with graded doses ofGFP-ADRP LDs (0, 10, 50, 100 :g/ml) for 24 h, and were subsequentlyanalyzed for SP-B mRNA expression (FIG. 3). Note the step-wise increasein SP-B mRNA expression over the dosage range used, resulting in an 80%increase at the highest LD dose (100:g/ml). Concomitant incubation withactinomycin D (FIG. 4) blocked the LD induction of SP-B mRNA expression.Using the same study design, we next examined the dose-dependent effectof GFP-ADRP LDs on SP-B protein expression by A549 cell monolayers (FIG.5). Here again, we observed a dose-dependent increase in SP-B content inresponse to LD exposure. Co-incubation of these LD-exposed cells withcycloheximide blocked the increase in SP-B protein expression (FIG. 6).

Uptake of ADRP-LDs Stimulates Surfactant Phospholipid Synthesis:

Incubation of A549 cells with 100 :g/ml of ADRP-LDs for 24 h, whichoptimally stimulated SP-B expression by these cells, stimulatedsaturated phosphatidylcholine synthesis 57-fold (FIG. 7). Co-incubationof A549 cells exposed to ADRP-LDs with graded doses of ADRP antibody(0.1, 0.4, 2 :g/ml) showed a dose-dependent inhibition ofADRP-LD-induced saturated phosphatidylcholine synthesis. Neitherpreimmune serum nor a non-specific IL-6 antibody showed inhibition ofthe ADRP-LD effect on saturated phosphatidylcholine synthesis,indicating the specificity of the ADRP antibody effect.

The Functional Effect of ADRP-LDs on SP-B Expression In Vivo:

In vivo administration of graded doses of ADRP-LDs (0, 45, 450, or 4500μg/kg) to ventilated adult rats (FIG. 8) resulted in a dose-dependentincrease in SP-B expression in the lung 30 minutes after administration.

Discussion

We have previously shown that cultured fetal rat lung fibroblasts takeup and store neutral lipid (Nunez and Torday (1995) J. Nutr. 125(6Suppl): 1639S-1644S; Rodriguez et al. (2001) Exp. Lung Res. 27(1):13-24; Torday et al. (1995) Biochim. Biophys. Acta., 1254(2):198-206;Torday and Rehan (2002) Am. J. Physiol. Lung Cell Mol. Physiol.283(1):L130-L135; Torday et al. (1998) Am. J. Med. Sci. 316(3): 205-208;Torday et al. (2001) Pediatr. Res. 49(6): 843-849), whereas culturedfetal rat ATII cells cannot (Torday et al. (1995) Biochim. Biophys.Acta., 1254(2):198-206); paradoxically, when these fibroblasts areco-cultured with ATII cells, neutral lipid is actively transferred fromfibroblasts to ATII cells and targeted specifically to synthesis ofsurfactant phospholipids (Id.), suggesting a “docking and trafficking”mechanism. We subsequently found that stretching ATII cells stimulatesprostaglandin E₂ production (Torday et al. (1998) Am. J. Physiol. 274(1Pt 1): L106-111), which subsequently stimulates the secretion of neutrallipid by fibroblasts, explaining why fibroblasts release neutral lipidin the presence (but not in the absence) of ATII cells in co-culture(Torday et al. (1995) Biochim. Biophys. Acta., 1254(2):198-206). But themechanism of lipid uptake and targeting remained unexplained. Thedemonstration of uptake of neutral lipid coated with ADRP in previousexperiments provided an explanation for why processing of neutral lipidby fibroblasts is necessary for this mechanism of neutral lipidtrafficking (Schultz et al. (2002) Am. J. Physiol. Lung Cell Mol.Physiol. 283(2): L288-1296). The current set of experiments confirmsthat ADRP is taken up by ATII cells, and now demonstrates that it istranslocated to the perinucleus, coordinately stimulating bothsurfactant phospholipid and protein synthesis.

The mRNA expression of ADRP, the neutral lipid droplet-associatedprotein in adult rodent lung, is second only to that in adipose tissue,the tissue that stores the largest amount of neutral lipid and exhibitsthe highest expression of ADRP mRNA (Brasaemle et al. (2000) J. Biol.Chem. 275(49): 38486-38493). In a previous study, we had found ADRPprotein expression in sections of rodent lung tissue localized aroundlipid droplets. We also reported that ADRP was developmentally expressedin fetal and newborn rat lung, paralleling the accumulation of neutrallipid in lung tissue. Furthermore, the ADRP expression was localized toLIFs, the interstitial lung fibroblasts characterized by cytoplasmicneutral lipid droplets (Londos et al. (1995) Biochem. Soc. Trans.,23(3): 611-615). In contrast, we found minimal expression of ADRP inprimary fetal rat ATII cells, the pneumocytes that lie adjacent to LIFsin the alveolar interstitium (Schultz et al. (2002) Am. J. Physiol. LungCell Mol. Physiol. 283(2): L288-1296) and are the sites of pulmonarysurfactant synthesis.

The present series of experiments validates the previous study showingADRP binding and translocation of ADRP-associated LDs to ATIIs (Id.);translocation is now expanded to the A549 cell perinucleus, where ADRPapparently quantitatively stimulates both de novo SP-B expression andsurfactant phospholipid synthesis.

During rat lung development LIFs lie in close apposition to ATIIs (Id.),and play a central role in the growth (Weaver et al. (2002) Semin. CellDev. Biol. 13(4): 263-270), differentiation (Shannon and Hyatt (2004)Annu. Rev. Physiol., 66: 625-645) and stability of the epithelial ATIIcell phenotype (Shannon et al. (2001) Am. J. Respir. Cell Mol. Biol.24(3):235-244). LIFs first appear during the canalicular phase of fetalrat lung development, and triacylglycerol content is maximal just beforethe appearance of surfactant-containing lamellar bodies in neighboringATII cells (Torday et al. (1995) Biochim Biophys Acta.,1254(2):198-206). Despite such apparent evidence for a precursor-productrelationship between fibroblast triacylglycerols and ATII cellsurfactant phospholipids, there was no empiric evidence for theexistence of such a mechanism until we (Id.) demonstrated thattriacylglycerols of fibroblast origin are specifically and activelyrecruited (Schultz et al. (2002) Am. J. Physiol. Lung. Cell Mol.Physiol. 283(2): L288-1296; Torday and Rehan (2002) Am. J. Physiol. LungCell Mol. Physiol. 283(1):L130-L135; Torday et al. (1998) Am. J. Med.Sci. 316(3): 205-208; Torday et al. (1998) Am. J. Physiol. 274(1 Pt 1):L106-111) for surfactant phospholipid synthesis by ATII cells inculture.

Initially, we (Torday et al. (1995) Biochim. Biophys. Acta.,1254(2):198-206) had demonstrated the accumulation of triacylglycerol bythe developing fetal rat lung fibroblast, increasing four- to five foldbetween embryonic days e18 and e22, with no increase in ATII celltriacylglycerol content. It was later revealed that isolated fetal ratlung fibroblasts, but not ATII cells, actively take up lipid and packageit into triacylglycerol-ADRP complexes, providing a mechanisticexplanation for the observed accumulation of triacylglycerols byfibroblasts, but not by ATII cells in vivo. To determine the mechanisticsignificance of these observations (Id.), we ‘loaded’ the fibroblastswith radiolabeled triacylglycerol and recombined them with ATII cells inorganotypic co-culture (Id.) to evaluate transit and metabolism offibroblast triacylglycerol by ATII cells. There was quantitativetransfer of triacylglycerol from fibroblasts to ATII cells, resulting ina three-fold increase in the satPC content of the ATII cells. To comparethe rate of satPC synthesis from fibroblast triglyceride to that due tocirculating substrate, the rate of fibroblast [³H]triacylglycerolincorporation into ATII cell phospholipids was simultaneously comparedto the rate of incorporation of extracellular [¹⁴C]glucose. Bothtriacylglycerol and glucose were incorporated into ATII cellphospholipids, particularly satPC and phosphatidylglycerol, which arethe principal surfactant phospholipids. The rate of triacylglycerolincorporation into satPC and phosphatidylglycerol was 10- to 23-foldhigher, respectively, than that of glucose. These data suggested theexistence of a specific mechanism for the shuttling of triacylglycerolfrom the LIF to the ATII cell.

A subsequent series of immunofluorescence experiments (Schultz et al.(2002) Am J Physiol Lung Cell Mol Physiol. 283(2): L288-1296) showedthat minimal expression of ADRP in ATII cells before co-culture withLIFs was greatly increased along with the transfer of lipid from LIFsafter co-culture with LIFs. In addition, anti-ADRP antibodies blockedthe transfer of lipid in the co-culture system. Both of theseobservations suggested an important role of ADRP in the mobilization ofintracellular lipid stores from LIFs to ATII cells during fetal lungmaturation. In this context, ADRP has also been found on the surface oflipid globules secreted by mammary epithelial cells (Heid et al. (1996)Biochem J., 320 (Pt 3):1025-1030). However, in isolated cultures of LIFsand LIFs co-cultured with ATII cells, we found no evidence of secretedADRP, by either Western blotting or radiolabeled protein techniques.During the time of increased triacylglycerol accumulation in thedeveloping lung, cytoplasmic projections are present between LIFs andATII cells (Adamson et al. (1991) Exp. Lung Res. 17(4): 821-835), givingrise to the possibility that the lipid transfer may occur via theseconnections.

In the present series of experiments, we have used a GFP-ADRP fusionconstruct to determine if ADRP is taken up by ATII cells and what itsintracellular fate is. We have discovered that ADRP-LD complexestraverse the plasma membrane and initially localize around the nucleus,subsequently migrating to the cytoplasm. This process is associated withup-regulation of both surfactant protein and phospholipid synthesis, andcan be blocked by inhibitors of RNA and protein synthesis. Takentogether, these data, for the first time, provide a cell/molecularmechanism for the long-recognized (Jobe and Ikegami (2001) Clin.Perinatol., 28(3): 655-669) coordinate regulation of the phospholipidand protein moieties of the surfactant. Of equal, if not greaterimportance is the fact that ADRP is regulated by ParathyroidHormone-related Protein (PTHrP) (Torday and Rehan (2002) Am. J. Physiol.Lung Cell Mol. Physiol. 283(1):L130-L135), linking the regulation ofsurfactant protein and phospholipid synthesis to stretch, since PTHrP isa stretch-regulated gene expressed by the ATII cell (Torday and Rehan(2002) Am. J. Physiol. Lung Cell. Mol. Physiol. 283(1):L130-L135; Tordayet al. (1998) Am. J. Med. Sci. 316(3): 205-208).

McGowan et al. (McGowan et al. (2001) Exp. Lung Res., 27(1): 47-63) haveevaluated the roles of lipoprotein receptors and Apolipoprotein E (ApoE)in the accumulation of circulating lipoproteins by LIFs. Because theyfound no correlation between developmental age of the LIFs and theirlipoprotein receptors or ApoE expression, they concluded that suchchanges must, alternatively, be due to the amounts of lipoprotein incirculation. The concentration of triglyceride in fetal rat circulationis 40-fold lower than in fetal rat lung LIFs (Torday et al. (1995)Biochim. Biophys. Acta., 1254(2):198-206), and although it increases inthe postnatal period, it does not correlate with the pattern oftriglyceride content in developing LIFs (Id.). Furthermore, we haveshown that both endocrine hormones (Nunez and Torday (1995) J. Nutr.125(6 Suppl): 1639S-1644S; Rodriguez et al. (2001) Exp. Lung Res. 27(1):13-24; Torday et al. (2001) Pediatr. Res. 49(6): 843-849) and paracrinefactors (Torday and Rehan (2002) Am. J. Physiol. Lung Cell Mol. Physiol.283(1):L130-L135; Torday et al. (1998) Am J Med. Sci. 316(3): 205-208)have direct effects on the rate of LIF triglyceride accumulation,indicating that this process is regulated at the cellular level. Incontrast to the dissociation of circulating triglyceride levels duringthe perinatal period from the ontogeny of triglycerides in LIFs, thepattern of LIF expression of ADRP (Schultz et al. (2002) Am. J. Physiol.Lung Cell Mol. Physiol. 283(2): L288-1296) is consistent with itshypothesized role in LIF triglyceride accumulation.

Lung surfactant production is widely recognized to be under bothhormonal (Mendelson (2000) Pp. 141-159 In: Endocrinology of the Lung.Humana Press, Totawa, N.J.) and paracrine regulation (Wolins et al.(2001) J. Biol. Chem. 276(7): 5101-5118). In the LIF-ATII cellco-culture system, dexamethasone was shown to selectively stimulate LIFtriacylglycerol incorporation into ATII cell satPC (Nunez and Torday(1995) J. Nutr. 125(6 Suppl): 1639S-1644S), indicating the existence ofa specific mechanism for triacylglycerol mobilization from thefibroblast to the ATII cells that is hormonally regulated. Studiesindicate that the increase in ADRP expression by ATII cells afterco-culture with LIFs is blocked by incubation with an antagonist ofPTHrP (37), a necessary determinant of lung maturation (Rubin et al.(2004) Dev. Dyn. 230(2): 278-289). That finding suggests that endogenousPTHrP promotes the lipid transfer between LIFs and ATII cells and thechange in ADRP expression in ATII cells that accompanies this transfer.

PTHrP is a stretch-regulated product of the ATII cell which signals theup-regulation of both ADRP (Torday and Rehan (2002) Am. J. Physiol. LungCell Mol. Physiol. 283(1):L130-L135) and leptin (Id.) by LIFs. Whenthese previous observations are combined with the present resultsshowing coordinate surfactant protein and phospholipid expression byATIIs through the action of ADRP-LDs, it provides the first integratedcell-molecular mechanism for the ‘on-demand’ stretch-regulatedsurfactant production, initially demonstrated by Faridy (Faridy (1976)Respir. Physiol. 27(1): 99-114), and then by others (Nicholas et al. J.Appl. Physiol. 53(6): 1521-1528).

The possible involvement of ADRP, a protein intrinsic to intracellularlipid droplets, in the transfer of triacylglycerol from LIFs to ATIIcells suggests a novel mechanism for the trafficking of neutral lipidsbetween these two cell-types. No ADRP protein was found in the culturemedium of LIFs alone or in co-cultures with ATII cells. Thisobservation, in combination with the close apposition (Gewolb and Torday(1995) Lab Invest. 73(1): 59-63) and cellular projections between LIFsand ATII cells (Adamson et al. (1991) Exp. Lung Res. 17(4): 821-835),would suggest that the lipid shuttling mechanism between these twocell-types is not the same as that for circulating lipoproteins, whichinvolves secretion and possible uptake of whole lipid particles. In thiscontext, TIP47, a recently described protein highly homologous to ADRP(Wright and Clements (1987) Am Rev. Respir. Dis. 136(2): 426-444), hasbeen reported to bind to the cytoplasmic domain of mannose 6-phosphatereceptors and mediate receptor uptake and targeting to the lysosomalcompartment. This pathway is very similar to the processing of lipidsand proteins for surfactant phospholipid synthesis and storage withinlamellar bodies, which are modified lysosomes (Whitsett and Glasser(1998) Biochim. Biophys. Acta. 1408(2-3): 303-311). Interestingly, aseparate study has also demonstrated that TIP47 targets to lipid storagedroplets (Miura et al. (2002) J. Biol. Chem. 277(35): 32253-32257).Furthermore, we have previously shown that LIFs secrete lipid and thatprostaglandin E₂ of ATII cell origin is an agonist for such secretion(Torday et al. (1998) Am. J. Physiol. 274(1 Pt 1): L106-111).

In conclusion, we provide a Schematic (FIG. 9) for this lipidtrafficking mechanism which depicts (1) the active recruitment ofcirculating lipid by the LIF, (2) the formation of ADRP-lipid dropletsby the LIF, (3) stimulation of ADRP-LD synthesis and secretion (4) inresponse to ATII-produced ADRP and prostaglandin E₂ (PGE₂),respectively, (5) ADRP-LD uptake by AIIs via a receptor-mediatedmechanism, and (6) coordinate stimulation of surfactant phospholipid andSP-B, resulting in simultaneous increases in surfactant protein andphospholipid production by the ATII cell.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of inhibiting the growth or proliferation of a tumor cell,said method comprising contacting said tumor cell with contacting saidtumor cell with a composition comprising an adipocytedifferentiation-related protein (ADRP) covalently coupled to, orcomplexed with, a lipid or liposome, wherein said lipid is complexedwith, or said liposome contains, a cancer therapeutic.
 2. The method ofclaim 1, wherein said cancer therapeutic is selected from the groupconsisting of retinoic acid, a retinoic acid derivative, doxirubicin,vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin,5-fluorouracil, a camptothecin derivative, interferon, tamoxifen, andtaxol.
 3. The method of claim 1, wherein said ADRP is a carboxylterminal fragment of ADRP of sufficient length to induce transport ofsaid lipid or liposome into an epithelial cell.
 4. The method of claim1, wherein said ADRP is a full length ADRP.
 5. The method of claim 1,wherein said cancer comprises a small cell carcinoma.
 6. The method ofclaim 1, wherein said lipid or liposome is a neutral lipid or a liposomeformed of neutral lipids.
 7. The method of claim 1, wherein said lipidor liposome is a neutral lipid or a liposome formed of neutral lipidscomprising triacylglycerol.
 8. The method of claim 1, wherein said lipidor liposome is a multilamellar liposome.
 9. The method of claim 1,wherein said lipid or liposome is a unilamellar liposome.
 10. A methodof preferentially delivering an effector to a tumor cell in a mammal,said method comprising: providing said effector in a liposome orcomplexed with a lipid, wherein said lipid or said liposome arecomplexed with or covalently attached to an adipocytedifferentiation-related protein (ADRP); administering said lipid orliposome to said mammal whereby said lipid or liposome is preferentiallyinternalized by said tumor cell.
 11. The method of claim 10, whereinsaid ADRP is a full length ADRP.
 12. The method of claim 10, whereinsaid lipid or liposome is a neutral lipid or a liposome formed ofneutral lipids.
 13. The method of claim 10, wherein said lipid orliposome is a neutral lipid or a liposome formed of neutral lipidscomprising triacylglycerol.
 14. The method of claim 10, wherein saidlipid or liposome comprises an agent selected from the group consistingof a retinoid, a prostanoids, an anti-inflammatories, a growth factor, athiazolidinediones, a chemokine, and a chemotherapeutic.
 15. The methodof claim 10, wherein said lipid or liposome is a multilamellar liposome.16. The method of claim 10, wherein said lipid or liposome is aunilamellar liposome.
 17. The method of claim 10, wherein said mammal isa human.
 18. The method of claim 10, wherein said tumor cell comprises asmall cell carcinoma.
 19. The method of claim 10, wherein said effectoris selected from the group consisting of a label, a cytotoxin, a drug, aprodrug, and a cytokine.
 20. The method of claim 10, wherein saideffector is a cytotoxin selected from the group consisting of aDiphtheria toxin, a Pseudomonas exotoxin, a ricin, an abrin, and athymidine kinase.
 21. The method of claim 10, wherein said effector is adetectable label selected from the group consisting of a radioactivelabel, a spin label, a colorimetric label, a fluorescent label, and aradio-opaque label.
 22. The method of claim 10, wherein said effectorcomprises an isotope selected from the group consisting of ⁹⁹Tc, ²⁰³Pb,⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁵²Fe, ⁵²Mn, ⁵¹Cr,¹⁸⁶, Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr,¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho,¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and ¹¹¹Ag.
 23. The method of claim10, wherein said effector comprises an alpha emitter.
 24. The method ofclaim 10, wherein said effector is a drug selected from the groupconsisting of retinoic acid, a retinoic acid derivative, doxirubicin,vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin,5-fluorouracil, a camptothecin derivative, interferon, tamoxifen, andtaxol.